Previous reports at the Annual Citrus Processors' Meetings 1960 and 1961
contained definite identifications of a number of carbonyl'components found in
orange essence, but only two flavor alcohols were definitely identified. Con-
sequently, work during the past year has been directed toward finding supple-
mentary techniques for identifying the gas chromatographically separated
alcohols so as to render a more meaningful interpretation to the gas chromato-
graphic results.

A procedure was described at the 1961 meeting for the paper chromatographic
analysis of alcohols as their phenyl- and naphthylurethan derivatives, Since
that time the use of the latter derivatives has been discontinued, and the o-,
m-, and p-nitrophenyl- and p-phenylazophenylurethan derivatives substituted
because of their superior physical properties and low limit of detection on the
paper chromatograms (Anal. Chem. 3h, 671 (1962). Spots of less than 1 pg. of
these materials can be located by illuminating the papers with ultraviolet
light, whereas a cumbersome spray procedure requiring 20 pg was necessary for
the phenyl- and naphthylurethans. The short chain alcohols, C1 C8, were best
separated as their m- or p-nitrophenylurethans but the p-phenylazophenylurethans
could also be used in some cases. The following Rf values were obtained using
Whatman No. 3MM paper dipped in 80-20 (v/v), methanol- propylene glycol. Hep-
tane saturated with methanol was the developing solvent.

Longer chain aliphatic alcohols and terpene alcohols were best determined
as their o-nitrophenylurethan derivatives, but the p-phenylazophenylurethans
could again be used. Some typical Rf values obtained for aliphatic alcohols on
Whatman No. 3MM paper dipped in 7% vaseline-heptane solution, and using 95-6
(v/v), methanol-water, as the developing solvent are as follows:

The paper chromatographic procedure was adapted to the study of orange
essence components as follows: first, the organic layer obtained by extracting
aqueous essence was treated by either the bisulfite technique or the Girard T
reagent to remove carbonyl components aldehydess and ketones). The carbonyl-
free essence extract so obtained was then passed through a column of either

activated alumina or silicic acid, using elution with progressively more polar
solvents to further separate the alcohols from the hydrocarbons and esters. The
concentrated alcohols were either reacted directly with the proper isocyanate
and the urethans paper chromatographed, or else the alcohols were first separated
gas chromatographically and the condensed components corresponding to the indi-
vidual peaks reacted with the isocyanate and the urethans chromatographed. Due
to the complexity of the mixture, the latter procedure was most satisfactory as
it permitted a cleaner separation than could be obtained with paper chromato-
graphy alone. The use of paper chromatographic procedure has been found superior
to infrared analysis because mixtures of components and compounds present in too
small a quantity to give a good spectrum will yield satisfactory data. Further,
in the case of the major essence alcohols it has been possible to obtain
sufficient amounts of the derivatives for melting point determinations. The p-
phenylazophenylurethans are superior for this purpose.

Using various combinations of the above mentioned techniques, the following
alcohols obtained from orange essence have been identified; also, listed are the
relative importance figures obtained from average peak amplitudes of the gas
chromatograms and these are only very rough estimates.

At the 1961 meeting some preliminary data were presented which indicated the
presence of acetic, propionic, and butyric acids in aqueous orange essence. Dur-
ing the past year further experiments have been conducted and the presence of
acetic and butyric acids definitely confirmed. They were accompanied by other
acidic materials not definitely identified. The, acids were determined by paper
chromatography as their p-phenylazophenacyl esters and as their ammonium salts.
In utilizing the esters, 2 liters of aqueous essence were made basic with dilute

NaOH and evaporated to dryness. The soluble portion of the residue was dissolved
in 10 ml of water and made acidic to litmus with dilute HC1. Then 10 ml of
ethanol and 1 g p-phenylazophenacyl bromide were added. The resulting solution
was refluxed, using a boiling chip, for 2 1/2 hours, cooled, and filtered to
obtain the insoluble crystals. These crystalline substances were dissolved in
Skellysolve B and separated into 7 crude fractions by passing through a silicic
acid column using Skellysolve B and benzene as the eluting solvents. The first,
second, and third fractions contained only impurities while the fourth through
seventh cuts contained p-phenylazophenacyl esters contaminated with p-phenylazo-
phenacyl alcohol, formed by hydrolysis of the bromide. The alcohol was removed
by passing through an alumina column. The crystals from fractions 4 and 5-were
then analyzed paper chromatographically using Whatman No. 3MM paper treated with
vaseline and 90-11 (v/v), methanol-water, as the developing solvent. Three
spots were obtained, the Rf values of which were 0.72, 0.52, and 0.33. These
corresponded to the esters of butyric, caprylic, and capric acids; however, an
infrared spectrum of the mixture indicated that at least one of these spots was
a non-ester impurity. Fractions 6 and 7 were shown paper chromatographically to
be a mixture of the acetic and butyric acid esters.

For the preparation of the ammonium salts, a liter of aqueous essence was
made basic with dilute NHO4H and evaporated on the steam bath. Small amounts of
NH40H were added during the evaporation to maintain basicity. The residue was
dissolved in 20 ml dilute NH40H and filtered. Chromatography of this material
on SS 589 acid-washed paper using propanol-ammonium hydroxide, 70-30 (v/v), as
developer and bromophenol blue as spray reagent revealed spots of Rf values
0.93, 0.73, 0.53, 0.18, plus indications of a spot at 0.13. The spots at 0.73
and 0.53 corresponded to butyric and acetic acids, respectively. A known mix-
ture of capric and caprylic acids had an Rf of 0.84, which did not correspond to
the highest Rf spot in the unknown, leaving the presence of these acids uncon-
firmed. Spraying of the chromatograms with ninhydrin showed ninhydrin sensitive
materials of Rf values 0.h3, 0.33, and 0.13. The upper spot could conceivably
be pyroglutamic acid (pyrolidone carboxylic acid), approximately Rf 0.1, which
has been reported in processed tomatoes (Food Research, 19, 106-11l (195).

To determine if any of the unknown acids belonged in the non-volatile
category, 500 ml of aqueous essence was evaporated to dryness without adding
base. The residue was dissolved in 10 ml distilled water and chromatographed
using propanol-ammonium hydroxide as described. The acetic and butyric acid
spots were almost indistinct, but the Rf 0.18 and 0.92 spots were more distinct
indicating that they were produced by non-volatile acids.

In addition to the identification of some alcohols and acids in orange
essence, gas chromatographic retention temperatures of several known esters
and terpene hydrocarbons were determined. These were compared with retention
temperatures of unknown component peaks found in the ester-hydrocarbon rich
cuts obtained by passing carbonyl-free essence extract through the alumina
column. Those knowns, coinciding most closely to unknown peaks in retention
temperature, were further studied by the enrichment procedure using one
column, namely Carbowax 20M. The esters checking by these procedures and